Too much light can be lethal for photosynthetic organisms. Under such conditionsharmful reactive oxygen species are generated at the reaction center level. Cyanobacteria havedeveloped photoprotective mechanisms to avoid this. One of them relies on the solubleOrange Carotenoid Protein (OCP) that binds a ketocarotenoid (hydroxyechinenone, hECN).Under strong blue-green illumination, OCP gets photoconverted from an orange inactive form(OCPo) to a red active one (OCPr). OCPr interacts with phycobilisomes, the majorcyanobacterial light harvesting antennae, and triggers heat dissipation of the excess lightenergy collected by these gigantic pigment-protein complexes. Consequently, excitationpressure on reaction centers and fluorescence emission decrease.OCPr binds to phycobilisome cores, containing mainly chromophorylated proteins ofthe allophycocyanin (APC) family. I constructed Synechocystis PCC 6803 mutants affected insome minor APC forms (ApcD, ApcF and ApcE). These special APCs play the role ofterminal emitters, i.e. funnel light energy to Chlorophyll a. Strong-blue green illuminationtriggered normal OCP-related fluorescence quenching in all mutant cells. The fluorescencedecrease induced by Synechocystis OCP in vitro was similar when using phycobilisomesisolated from wild-type or mutant cells. These results demonstrated that the terminal emittersare not needed for interaction with the OCP and they strongly suggested that OCPr interactswith one of the major APC forms of the phycobilisome core.Phycobilisomes containing 2, 3 or 5 APC cylinders per core were isolated fromdifferent cyanobacterial strains. Synechocystis and Arthrospira OCPs were purified from overexpressingSynechocystis mutant strains. I then performed in vitro OCP/phycobilisomeinteraction studies. The number of APC cylinders per core had no clear influence on theamount of fluorescence quenching. Both OCPs behaved very differently, one appearing muchmore species-specific than the other. Structure-based hypotheses were emitted to explain suchdissimilarity.Arthrospira OCP N-terminal and C-terminal domains were separated throughproteolysis. The isolated N-terminal domain retained a bound carotenoid, which displayedsimilar conformation than in OCPr. This isolated N-terminal domain triggered importantphycobilisome fluorescence quenching even under dark conditions. In contrast, the isolated Cterminaldomain attached no pigment and had no visible effect on phycobilisome emission. Itwas then proposed that only the N-terminal domain of OCP is implied in interactions withphycobilisomes. The C-terminal domain modulates its activity.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Plant carotenoid cleavage dioxygenases (CCD) are a family of enzymes that catalyse the oxidative cleavage of carotenoids and/or apocarotenoids. Carotenoids are synthesised in plastids (primarily chloroplasts and chromoplasts), where they are involved in light-harvesting and protecting the photosynthetic apparatus from photo-oxidation. The carotenoid-derived apocarotenoids fulfil a number of roles in plants such as phytohormones, pollinator attractants and flavour and aroma compounds. Due to the floral and fruity characteristics that apocarotenoids contribute to wine, these C13 compounds have received interest in grapevine (Vitis vinifera L.). The CCD gene family in Arabidopsis consists of nine members, all encoding for enzymes that catalyse the cleavage of carotenoids. The enzymes in this family include 9-cis-epoxydioxygenases (NCEDs) and four classes of CCD. NCEDs and CCD7 and CCD8 are involved with plant hormone synthesis, e.g. abscisic acid (ABA) through cleavage by NCED and strigolactone (SL) through the sequential cleavage of carotenoids by CCD7 and CCD8, respectively. SLs are a fairly new class of plant hormone which are involved in several aspects of plant growth and development. The most extensively characterised role of SLs is their involvement in the inhibition of shoot-branching. CCD1 and CCD4 cleave a variety of carotenoids to form pigments and aroma compounds. For example, CCD1 forms β-ionone and β-damascenone, which are important varietal flavours of wine, and CCD4 is involved in synthesis of the pigment and aroma compounds of saffron and annatto. CCD1 enzymes symmetrically cleave the 9,10 (9’,10’) double bonds of multiple carotenoids to produce a C14 dialdehyde and two C13 products. Additional CCD1 cleavage activity at 5,6 (5’,6’) double bonds of lycopene has been reported. Previous studies have shown that CCD1 isolated from V. vinifera (VvCCD1) was able to cleave multiple carotenoid substrates in vitro, namely zeaxanthin, lutein and β-carotene at 9,10 (9’,10’) double bonds and both the 5,6 (5’,6’) and 9,10 (9’,10’) double bonds of lycopene. None of the other VvCCDs, except VvCCD4a have been isolated (but no functionality was illustrated) and characterised yet. CCD4 enzymes also cleave carotenoids at the 9,10 (9’,10’) double bond positions. The presence of plastid-target peptides implies that the CCD4 enzymes have continuous access to carotenoids. Therefore it is suggested that CCD4s are responsible for carotenoid maintenance, where CCD1s contribute towards volatile production. To test this hypothesis VvCCD1, VvCCD4a and VvCCD4b were isolated from V. vinifera (cv Pinotage) cDNA and cloned into a pTWIN1 protein expression vector. Substrate specificity of each VvCCD was tested by co-transforming a carotenoid accumulating E. coli strain with a CCD expression vector. Carotenoids synthesized by the bacteria were identified and quantified by UPLC-analysis, while the concentration of the apocarotenoids, were measured in the headspace of the bacterial cultures using HS-SPME-GC-MS. Several optimisations were done to minimize the natural degradation of the carotenoids; to ensure that the apocarotenoid formation is predominantly due to the enzymatic cleavage by the VvCCDs and not due to oxidation or other non-enzymatic degradation. The HS-SPME-GC-MS analysis indicated that all isoforms cleaved phytoene, lycopene and ε-carotene. Additionally VvCCD1 cleaved a carotenoid involved in photosynthesis, namely β-carotene, while VvCCD4a cleaves neurosporene and VvCCD4b cleaves neurosporene and ζ-carotene, carotenoids not involved in photosynthesis. This study has illustrated that VvCCD1 cleave carotenoids necessary for photosynthesis and VvCCD4s cleave carotenoids which were not present in berry tissue, suggesting their role in carotenoid maintenance. Therefore in planta substrates for CCD1 could possibly be C27 apocarotenoids generated from enzymatic cleavage through CCD4 (role in carotenoid maintenance), CCD7 and/or photo-oxidation, which are then transported from the plastid to the cytosol or possibly C40 carotenoids that are released during senescence or when the plastid membrane is damaged, thus releasing important aroma compounds. Thus the identification of the in vivo substrates has contributed to the understanding the in planta functions of these enzymes / AFRIKAANSE OPSOMMING: Die plant ensiemfamilie van karotenoïedsplitsingdioksigenases (CCDs) kataliseer die oksidatiewe splitsing van karotenoïede en/of apokarotenoïede. Karotenoïede word in plastiede (primêr chloroplaste en chromoplaste) sintetiseer en is betrokke by lig-absorpsie en die beskerming van die fotosintetiese apparaat teen foto-oksidasie. Die apokarotenïede afkomstig van karotenoïede dien onder meer as planthormone, geur- en aromakomponente en om bestuiwers aan te lok. Aangesien apokarotenoïede bydra tot die vrug- en blomgeure van wyn is die C13-verbindings binne wingerd (Vitis vinifera L.) van belang. Al nege lede van die CCD geenfamilie in Arabidopsis kodeer karotenoïedsplitsingsensieme. Die ensiemfamilie sluit 9-sis-epoksidioksigenases (NCEDs), en vier klasse CCD in. NCEDs en CCD7 en 8 is betrokke by die sintese van planthormone, naamlik absissiensuur (ABA) deur NCED en strigolaktone (SL) deur die opeenvolgende aksie van onderskeidelik CCD7 en CCD8. SLe is redelik onlangs as planthormone indentifiseer en is betrokke by ‘n verskeie aspekte van die groei en ontwikkeling van plante. Die rol van SL in inhibisie van vertakking is die beste gekarakteriseerde van hierdie aspekte. CCD1 en CCD4 splits ‘n verskeidenheid karotenoïede om pigmente en aromakomponente te vorm. CCD1 vorm byvoorbeeld β-jonoon en β-damasenoon, beide belangrike kultivar-spesifieke wyngeure. CCD4 vorm weer die pigment en aromakomponente van saffraan en annatto. Die CCD1 ensieme splits die 9,10 (9’,10’) dubbelbindingsetels van verskeie karotenoïede simmetries en vorm een C14-dialdehied en twee C13-produkte. Daar is voorheen melding gemaak van verdere splitsing deur CCD1 by die 5,6 (5’,6’) dubbelbindingsetels van likopeen. Vroeër is getoon dat die CCD1 isovorm wat uit V. vinifera geïsoleer is, naamlik VvCCD1, in vitro seaxantin, luteïen en β-karoteen by die 9,10 (9’,10’) dubbelbindingsetels kon splits, en likopeen by beide die 9,10 (9’,10’) en 5,6 (5’,6’) dubbelbindingsetels. Geen ander VvCCDs is al isoleer en funksioneel gekarakteriseer. VvCCD4a is isoleer, maar geen funksie is bepaal nie. CCD4 ensieme splits ook die 9,10 (9’,10’) dubbelbindingsetels van karotenoïede. Aangesien CCD4 ensieme ‘n plastied-bestemmingspeptied besit behoort dié ensieme konstant toegang tot karotenoïede te hê, wat dui op hul rol in die handhawing van die karotenoïedbalans, terwyl CCD1-ensieme bydra tot die sintese van vlugtige verbindings. Om hierdie hipotese te toets is VvCCD1, VvCCD4a en VvCCD4b uit V. vinifera (kv Pinotage) kDNS isoleer in binne ‘n pTWIN1 proteïenuitdrukkingsvektor kloneer. Die substraatspesifisiteit van elke VvCCD is getoets deur ‘n karotenoïedakkumulerende E. coil stam te transvormeer met ‘n CCD-uitdrukkingsvektor. UPLC-analise is gebruik om karotenoïede wat deur die bakterium sintetiseer is te kwantifiseer en identifiseer, terwyl die apokarotenoïedinhoud en -konsentrasie van die boruimte van die bakteriële kultuur met HS-SPME-GC-MS bepaal is. Verskeie aspekte van die proses is optimaliseer om natuurlike afbreking van karotenoïede te minimeer. Daardeur is verseker dat die apokarotenoïedvorming primêr vanweë die ensiematiese splitsing deur VvCCDs plaasvind en nie deur oksidasie of ander nie-ensiematiese afbreking. Die HS-SPME-GC-MS metings het aangedui dat al drie isovorme fitoëen, likopeen en ε-karoteen kan splits. VvCCD1 kan daarby β-karoteen splits, terwyl VvCCD4a neurosporeen, en VvCCD4b neurosporeen en ζ-karoteen kan splits, beide karotene wat nie betrokke is by fotosintese nie. Dié studie toon dat VvCCD1 die karotenoïede splits wat benodig word vir fotosintese, terwyl beide VvCCD4 isovorme karotenoïede splits wat nie in druiwekorrels gevind word nie. Dit dui op hulle rol in die handhawing van karotenoïedpoele. Die in planta substrate vir CCD1 mag dus die C27-apokarotenoïede wees wat deur CCD4 (as deel van karotenoïedhandhawing), CCD7 en/of foto-oksidasie gevorm word en na die sitosol vervoer word, of moontlik die C40-karotenoïede wat tydens veroudering óf wanner die plastiedmembraan beskadig is in die sitosol vrygestel word. Die identifisering van die in vivo substrate het dus bygedra to die begrip van die in planta funksies van die ensieme.

PINHO, Erotéide Leite de. Extração de pigmentos carotenóides da carapaça do camarão e sua utilização em um produto derivado de pescado. 2001. 48 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Tecnologia de Alimentos, Fortaleza-CE, 2001 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-06-03T13:54:29Z No. of bitstreams: 1 2001_dis_elpinho.pdf: 379735 bytes, checksum: 56d523219601d0f6243134cbd8534ed3 (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-06-03T13:55:13Z (GMT) No. of bitstreams: 1 2001_dis_elpinho.pdf: 379735 bytes, checksum: 56d523219601d0f6243134cbd8534ed3 (MD5) / Made available in DSpace on 2016-06-03T13:55:13Z (GMT). No. of bitstreams: 1 2001_dis_elpinho.pdf: 379735 bytes, checksum: 56d523219601d0f6243134cbd8534ed3 (MD5) Previous issue date: 2001 / Waste material in the fish industry including the shells from the crustacean, constitute a very high percentage. These shells are rich in carotenoids pigments, which may have a high pigmentation value when used in foods. It is common in some countries the use of the extracted pigments in feeds.The aim of this experiment was to study the process of carotenoids extraction and to verify its pigmenting potential in a minced fish food product. The solvent extraction technique was used after testing other extraction procedures. Extracted pigments were characterized by spectrophotometry. Pigments were then included in the fish formulation. Fish products were packaged with and without vacuum and storage in the dark at –20ºC for 60 days. The color of the products was measured every 15 days with the CIE system which determines parameters L*, a* and b*. The spectrophotometry study showed products of degradation of astaxanthin in the shell extract. The luminosity (L*) of the color in products packaged under vacuum was lower (p<0.05) with 15 and 45 days of storage than in those stored without vacuum. Vacuum packaged products showed that with 0 days of storage the yellowness component (b*) was higher (p<0.05) in vacuum packaged products than in those stored without vacuum. The reverse occurring with 45 days of storage. After 60 days frozen storage vacuum packaging did not affect the color characteristics of the fish product. / O descarte na indústria de pescado, inclusive o de carapaças de crustáceos, constitui um percentual bastante elevado. Estas cascas são ricas em pigmentos carotenóides, os quais, apresentam um alto valor de pigmentação, sendo corrente em alguns países a extração e posterior utilização dos mesmos em rações. O objetivo deste experimento foi estudar a extração dos pigmentos carotenóides das cascas de camarão e verificar seu potencial de uso como aditivo natural de cor em um produto à base de pescado. Neste estudo foram desenvolvidos testes preliminares para escolha de um método para a extração dos pigmentos optando-se pela extração com solvente. Caracterizou-se ospigmentos extraídos das cascas de camarão e avaliou-se a influência do extrato pigmentado aplicado em um produto à base de pescado, o qual foi embalado à vácuo e sem vácuo e submetido à uma estocagem (-20ºC) durante um período de60 dias. A cor dos produtos foi medida a cada 15 dias no sistema CIE determinando-se os parâmetros de L*, a* e b*. A análise espectrofotométrica do extrato de camarão apresentou produtos de degradação da astaxantina. Os produtos pigmentados com o extrato de camarão e embalados à vácuo apresentaram valores de luminosidade (L*) menores (p < 0,05) que os embalados sem vácuo com 15 e 45 dias de armazenamento. Para o componente de intensidade de cor amarela (b*) os produtos embalados à vácuo apresentaram valores maiores (p<0,05) no início do armazenamento (0 dias) e menores (p<0,05) com 45 dias de armazenamento, em relação àqueles embalados sem vácuo. Contudo, no período de 60 dias de armazenamento não foi observado efeito significativo da embalagem à vácuo sobre as características de cor dos produtos.

abstract: The first chapter reviews three decades of artificial photosynthetic research conducted by the A. Moore, T. Moore, and D. Gust research group. Several carotenoid (Car) and tetrapyrrole containing molecules were synthesized and investigated for excitation energy transfer (EET), photoregulation, and photoprotective functions. These artificial photosynthetic compounds mimicked known processes and investigated proposed mechanisms in natural systems. This research leads to a greater understanding of photosynthesis and design concepts for organic based solar energy conversion devices. The second and third chapters analyze the triplet energy transfer in carotenoid containing dyads. Transient absorption, time-resolved FTIR and resonance Raman spectra revealed that in a 4-amide linked carotenophthalocyanine dyads the Car triplet state is shared across the larger conjugated system, which is similar to protein complexes in oxygenic photosynthetic organisms. In a carotenopurpurin dyad (CarPur) a methylene ester covalent bond prevents the purpurin (Pur) from influencing the Car triplet based on the transient absorption, time-resolved FTIR and resonance Raman spectra. Thus CarPur resembles the antenna proteins from anoxygenic photosynthetic bacteria. Additional examples of carotenoporphyrin dyads further demonstrates the need for orbital overlap for ultrafast triplet energy transfer and the formations of possible intramolecular charge transfer state. The fourth chapter studies a 4-amino phenyl carotenophthalocyanine and its model compounds using high temporal resolution transient absorption spectroscopy techniques. EET from the Car second excited (S2) state to the phthalocyanine (Pc) was determined to be 37% and a coupled hot ground state (S*)/Pc excited state spectrum was observed. Excitation of the tetrapyrrole portion of the dyad did not yield any kinetic differences, but there was an S* signal during the excited states of the dyad. This demonstrates the EET and photoregulating properties of this artificial photosynthetic compound are similar to those of natural photosynthesis. The last chapter covers the synthesis of silicon Pc (SiPc) dyes and the methods for attaching them to gold nanoparticles and flat gold surfaces. SiPc attached to patterned gold surfaces had unperturbed fluorescence, however the selectivity for the gold was low, so alternative materials are under investigation to improve the dye's selectivity for the gold surface. / Dissertation/Thesis / Ph.D. Chemistry 2014

Submitted by (edna.saturno@ufrpe.br) on 2016-07-26T12:47:23Z No. of bitstreams: 1 Cristiane Rodrigues de Araujo.pdf: 1357200 bytes, checksum: e679be2445ef965c1948578b245819fa (MD5) / Made available in DSpace on 2016-07-26T12:47:23Z (GMT). No. of bitstreams: 1 Cristiane Rodrigues de Araujo.pdf: 1357200 bytes, checksum: e679be2445ef965c1948578b245819fa (MD5) Previous issue date: 2012-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This work had as objective to characterize of freeze-dried bark of mango Tommy Atkins, come from organic farming, and defining the conditions of the polyphenols extraction process. The peels were crushed and the flour was submitted to the determination of proximate composition, minerals and bioactive phytochemicals and antioxidant potential employing techniques titrimetric, spectrophotometric and chromatographic. To define the optimal conditions of the process was applied the of factorial design 2² as independent variables: temperature 25-50° C and time stirring 30 to 90 min), and as response the total phenolics concentration in the extract. This experimental design was applied in the following process types: a) Extraction sequentially- hydroacetonic solution (80%), followed by hydromethanolic solution (80%); b) Extraction sequentially- hydromethanolic solution (80%), followed by hydroacetonic solution (80% ); c) Extraction non-sequential-hydroacetonic solution (80%) and d) extraction non-sequential- hydromethanolic solution (80%). The extracts were submitted to antioxidant assays, capture radicals in model system (DPPH, ABTS, ORAC) and antioxidant assays in lipid systems (coupled oxidation of β -carotene-linoleic acid, peroxidation of linoleic acid - ferric thiocyanate method, and accelerated storage test in an oven) and high performance liquid chromatography analyses for identification of polyphenols. The mango flour showed high levels of carbohydrates, potassium, sodium, calcium and phosphorus, total phenolics, ascorbic acid and β-carotene. The factorial design defined as the ideal sequential extraction (80% methanol, followed by 80% acetone) and the following process conditions: temperature 37.5°C and 25ºC; time stirring of 30 and 60 min, whose extracts showed, respectively, higher total phenolic content (2407.16μg. mL-1) (Ext-5), and increased sequestration capacity DPPH (95%) (Ext-7). Both extracts exhibited significant antioxidant capacity against free radicals ABTS, DPPH and ORAC, and in lipid system. The chromatographic analysis revealed the presence in both extracts of the phenolic acids (benzoic acid and cinnamic acid derivatives), flavonoids and a xanthone, but with quantitative and qualitative differences between the profiles of the extracts. Synaptic acid, syringic acid, taxofoline, isoquercetine and mangiferin were the major constituents of these extracts. Thus, considering the chemical composition and antioxidant capacity, the agro-industrial waste of Tommy Atkins mango emerges as a potential source of natural antioxidant. / Este trabalho teve como objetivo caracterizar cascas liofilizadas de manga Tommy Atkins, provenientes de um cultivo orgânico, quanto à composição centesimal, de minerais, de fitoquímicos bioativos, e o potencial antioxidante além de definir as condições ideais para obtenção de extratos com elevado teor de polifenóis. As cascas liofilizadas foram trituradas, e a farinha submetida às determinações analíticas empregando-se técnicas titulométricas, espectrofotométricas e cromatográficas. Para definição das condições ideais de processo (tempo de agitação; temperatura e tipo de processo) aplicou-se o planejamento fatorial 2² , tendo como variáveis independentes: temperatura de 25 a 50°C e tempo de agitação de 30 a 90 min; e como resposta o teor de fenólicos e a capacidade de sequestrar o radical DPPH. O planejamento foi aplicado aos seguintes tipos de processo: a) Extração sequencial-solução hidroacetônica (80%), seguida da solução hidrometanólica (80%); b) Extração sequencial-solução hidrometanólica (80%), seguida da solução hidroacetônica (80%); c) Extração não sequencial-solução hidroacetônica (80%); d) Extração não sequencial-solução hidrometanólica (80%). Os extratos obtidos utilizando as condições ideais foram submetidos aos ensaios antioxidantes, de captura de radicais, em sistema modelo (DPPH; ABTS; ORAC) e aos ensaios antioxidantes em sistemas lipídicos (oxidação acoplada do B-caroteno/ácido linoléico; da peroxidação do acido linoleico – método tiocianato férrico; e do teste acelerado em estufa), e a cromatografia liquida de alta eficiência (HPLC) para identificação dos polifenóis. A farinha de manga apresentou elevados teores de carboidratos, potássio, sódio, cálcio e fósforo, fenólicos totais, ácido ascórbico e β-caroteno. O planejamento fatorial definiu como ideal a extração sequencial (metanol 80%, seguido por acetona 80%) e as seguintes condições de processo: temperatura de 37,5ºC e 25ºC e tempo de extração de 30 e 60min; cujos extratos apresentaram, respectivamente, maior teor de fenólicos totais (2407,16μg. mL-1) (Ext-5), e maior capacidade de sequestro do radical DPPH (95%) (Ext-7). Os dois extratos exibiram expressiva capacidade antioxidante frente aos radicais livres ABTS, DPPH e ORAC; bem como em meio lipídico. A análise cromatográfica revelou a presença, nos dois extratos, de ácidos fenólicos (derivados do ácido benzóicos e cinâmicos), flavonóides e uma xantona, porém com diferenças quantitativas e qualitativas entre os perfis dos extratos. O ácido siríngico, ácido sináptico, a taxofolina, a isoquercetina e a mangiferina foram os constituintes majoritários nestes extratos. Assim, considerando a composição química e a ação antioxidante, o resíduo agroindustrial de manga Tommy Atkins surge como uma fonte potencial de antioxidante natural.

Lipid soluble carotenoids are micronutrients present in human milk that serve as precursors of vitamin A and also play an important role protecting cells from damage arising from photooxidative processes and reactive oxygen species. Fatty acids comprise about 3-5% of human milk and are mainly present as triglycerides. They are a major energy source for the infant and are necessary to support cell growth required for normal development and maturation of critical organs. Transport of carotenoids into milk has been little studied and there has been no previous investigation of the relationship of carotenoid transport with that of individual fatty acid secretion into milk. In the present study, levels of the carotenoids, lutein, zeaxanthin, b-cryptoxanthin, and b-carotene, in maternal serum, infant cord blood, and milk obtained from 74 Peruvian mothers were measured by HPLC methods. The fat content and fatty acid profile of maternal milk were determined by GC-FID and confirmed by GC-MS. Twenty nine fatty acids were identified and quantified after conversion to methyl esters. Statistical analysis was employed to investigate potential trends and relationships among the carotenoids in all three fluids as well as between carotenoids and fatty acids present. Concentrations of lutein in maternal serum and milk as well as maternal serum and infant cord blood were highly correlated (r =0.43, p

Licopeno, um pigmento natural considerado o mais potente antioxidante dentre os carotenoides, é oque tem maior incidência no soro humano. Seu consumo regular está relacionado principalmente com a prevenção do câncer de próstata. Porém, estudos também demonstram sua relação com a prevenção de câncer de pâncreas e bexiga, doenças cardiovasculares como a aterosclerose e doenças neurodegenerativas. Todavia, por ser altamente insaturado o licopeno é susceptível à degradação, sendo degradado na presença de luz, oxigênio e se exposto a altas temperaturas. A microencapsulação entra como uma alternativa para tentar garantir maior estabilidade a este carotenoide. A técnica de spray-chilling, por dispensar o emprego de altas temperaturas e solventes durante o processo de atomização, representa uma alternativa promissora na encapsulação do licopeno. Os objetivos deste trabalho foram encapsular uma solução oleosa de licopeno (10%) através da técnica de spray-chilling,utilizando gordura vegetal low trans como carreador, caracterizar as micropartículas obtidas e avaliar a biodisponibilidade do licopeno livre e encapsulado em ratos wistars. Foram formulados seis tratamentos, que diferiam pela concentração de solução comercial de licopeno, sendo T1 com 20%, T2 com 23,1%, T3 com 28,6%, T4 com 33,3%, T5 com 17,9% mais 10% de goma arábica e T6 com 19,2% mais 5% de carboximetilcelulose (CMC). As micropartículas obtidas destes tratamentos foram avaliadas quanto a tamanho e distribuição, morfologia por microscopia eletrônica de varredura (MEV), espectroscopia de infravermelho com transformadas de Fourier (FT-IR), difração de raios-X (DRX). A estabilidade do licopeno encapsulado foi avaliada em diferentes condições de armazenamento (sob vácuo, umidade relativa de 33%, temperatura de refrigeração e ambiente) e também foi determinada por meio de quantificações periódicas de licopeno, bem como através da análise análise da cor instrumental. A biodisponibilidade foi avaliada utilizando-se 68 animais divididos em grupos, para os quais se administrou por gavagem o licopeno livre e o encapsulado. O tamanho das micropartículas obtidas ficou em torno de 60-110 &micro;m e a distribuição foi polidispersa, independente da concentração de licopeno. A microscopia revelou micropartículas esféricas, com superfície rugosa, com alguns poros e tamanhos variados. No FT-IR verificou-se que não houve formação de ligações distintas na solução oleosa de licopeno e nas amostras atomizadas. Nos difratogramas observou-se a presença da forma polimórfica &beta; para o agente carreador e para as micropartículas. Na estabilidade a adição da goma arábica e o armazenamento sob temperatura de refrigeração e vácuo, foram as melhores condições para retardar a degradação do licopeno. Os resultados dos ensaios de biodisponibilidade foram inconclusivos. Desta forma, conclui-se que é possível encapsular licopeno através da técnica de spray-chilling, porém, para trabalhos futuros, seriam necessários aprimoramentos na técnica de encapsulação e/ou na formulação para conferir maior proteção ao carotenoide, bem como adequações na metodologia para determinação de sua biodisponibilidade, para obtenção de resultados conclusivos. / Lycopene, a natural pigment considered the most potent antioxidant among the carotenoids, it has the higher incidence in the human serum. Its regular consumption is mainly related with the prevention of prostate cancer. However, studies also show its relation to the prevention of pancreatic cancer and bladder cancer, cardiovascular diseases such as atherosclerosis and neurodegenerative diseases. However, by being highly unsaturated the lycopene is susceptible to degradation, being degraded in the presence of light, oxygen and if exposed to high temperatures. The microencapsulation comes like an alternative to ensuring higher stability for this carotenoid. The technique of spray-chilling represents a promising alternative to encapsulation of lycopene. The aims of this study were to encapsulate an oily solution of lycopene (10%) through of the technique of spray-chilling, using a low-trans fat as carrier, to characterize the obtained microparticles and to evaluate the bioavailability of lycopene free and encapsulated in Wistar rats. Six treatments were formulated, that differed by the content of oily solution of lycopene:T1 with 20%, T2 with 23.1%, T3 with 28.6%, T3 with 28.6%, T4 with 33.3%, T5 with 17.9% plus 10% of Arabic gum and T6 with 19.2% plus 5% of carboxymethylcellulose (CMC). The microparticles obtained from these treatments were evaluated for size and distribution, morphology by scanning electron microscopy (SEM), infrared spectroscopy with Fourier transform (FT-IR) and X-ray difraction (XRD). The stability of the lycopene encapsulated was evaluated by its periodic quantification at different storage conditions (vacuum, relative humidity of 33%, refrigeration temperature and environment temperature). Instrumental color, \"L\" and \"a\" parameters, also was measured. The bioavailability was evaluated using 68 animals, for which the free and lycopene encapsulated were administered by gavage. The size of microparticles obtained was around 60-110 &micro;m and the distribution was polydisperse, independent of the concentration of lycopene. The microscopy revealed spherical microparticles, with rough surface, with some pores and varying sizes. In the FT-IR it was found that there was no formation of distinct bonds in oily solution of lycopeno and the atomized samples. In the diffraction patterns observed the presence of polymorphic form \"&beta;\" for the carrier agent and microparticles. On the stability the addition of Arabic gum and the storage at refrigerator temperature under vacuum, were the best conditions to delay the degradation of lycopene. The results of bioavailability assays were inconclusive. As conclusion, it is possible to encapsulate lycopene using the technique of spray-chilling but to future works, would be required improvements in the technique of encapsulation and/or formulations to give more protection to the carotenoid, as well as adjustments in the methodology for determination of their bioavailability, in order to obtaining conclusive results.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13346号 / 論農博第2889号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5253(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 菅原 達也, 教授 佐藤 健司, 教授 澤山 茂樹 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

This diploma thesis deals with the molecular characterization of carotenogenic yeasts. The techniques used for the analysis of the conserved regions of the D1/D2 rDNA region of the 26S ribosomal large subunit region and the ITS1 and 5,8-ITS2 regions were nested PCR and DGGE. The results of DGGE show that all analyzed yeast strains have very similar sequences of these regions The yeast Rhodotorula mucilaginosa with the collection number CCY 20-7-28 showed differences from the other carotenogenic yeast strains. As a part of melucular characterisation using ribosomal gene sequences, eight yeast strains were examinated for substrate utilisation tests using different substrates. Characterisation of growth and metabolite production was tested in each strain too. The next aim of this thesis was to prepare a carotenoid yeast strain characterized by overproduction of metabolites, in particular carotenoids and lipids,. Yeasts were subjected to a random mutation caused by UV irradiation and the influence of this mutantagen onthe production of metabolites was evaluated. As a candidate yeast strain C. capitatum CCY 10-1-2 was selected. This selection was based on previous studies due to its good production of lipids using waste glycerol as asubstrate. This strain was subsequently adapted to waste whey, glycerol, and a glucose as a basic carbon source.

Corynebacterium poinsettiae mutant strains blocked in carotenoid biosynthesis were obtained by treatment with the mutagen N-methyl-N1-nitro-N-nitrosoguanidine. Additional carotenoid (Crt) mutant strains were obtained from a previous study conducted in our laboratory. Fifty-nine Crt mutants affected in carotenoid biosynthesis were examined by a normal phase high performance liquid chromatography (HPLC) system. Pigment extracts of Crt mutants and C. poinsettiae wild type strains were resolved by an isocratic system with hexane:acetone:dicholoromethane, 11.35:1.73:1.00 (by vol.) as the eluting solvent. In addition to the five major peaks, twelve minor peaks were observed in the wild type C. poinsettiae strain used in this study. Crt mutant and wild type strain peak heights were measured from the individual chromatograms and the peak height data set created was analyzed using the Statistical Analysis System program to perform a cluster analysis. The cluster analysis revealed five carotenoid mutant groups. Carotenoid pigments which accumulated or were absent in each of the cluster groups are reported. Cluster group 1 mutants (CrtA) are blocked in the dehydrogenase(s) which is(are) responsible for the dehydrogenations between phytoene and lycopene. Cluster group 2 mutants (CrtB) appear to be blocked at a second dehydrogenase specific for the dehydrogenation from C.p. 470 to C.p. 496. Cluster group 3 mutants (CrtC) are blocked at a cyclization step in the pathway which involves cyclization of C.p. 496 to C.p. 470 and which may cyclize C.p. 473 to C.p. 450. The genes CrtA and CrtB map only 0.5 map units from each other while CrtA and CrtC map 2.1 map units from one another. Mutants which accumulate end products but which lack certain precursors indicate a branched pathway for pigment biosynthesis exists in this organism. Media for the formation, fusion and regeneration of C. poinsettiae protoplasts are reported and a protocol for the use of these media in genetic crosses of strains blocked in carotenoid biosynthesis is described. While isolating antibiotic resistant mutants useful in genetic analyses, novobiocin resistant mutants were observed to have a distinctly different colony pigment phenotype as compared to the wild type strain. HPLC chromatograms of a novobiocin resistant strain showed a distinctly different carotenoid pigment profile. The results provide evidence for differential gene expression in the carotenoid biosynthetic pathway when these mutants are grown in the presence of novobiocin.